The present invention relates to, in a multi-capillary electrophoretic apparatus comprising such a multi-capillary array migration part that a plurality of capillary columns are arranged so that samples are injected into the respective capillary columns and simultaneously electrophoresed in all capillary columns and an optical measuring part irradiating capillaries with light in the multi-capillary array migration part and measuring absorbance by the samples in the irradiated parts and fluorescence from the samples, a capillary cassette forming the multi-capillary array migration part.
Such a multi-capillary electrophoretic apparatus is used for separating/analyzing nucleic acid, protein, peptide, sugar and the like, and particularly plays an important role for analysis of the base sequence of DNA. The multi-capillary electrophoretic apparatus for sequence determination of DNA employs Sanger""s reaction, electrophoreses a DNA fragment (fragment) sample labeling a primer or a terminator with a fluorescent material and detects fluorescence from the DNA fragment sample during migration for deciding the base sequence.
For sequence determination of DNA having long base sequence such as a human genome, a DNA sequencer having high sensitivity, a high speed and large throughput is necessary. As one method thereof, a multi-capillary DNA sequencer arranging capillary columns charged with gels in plural is proposed in place of that employing flat plate type slab gels. In the capillary column, a sample is not only easy to handle or inject but also can be migrated at a high speed and detected in high sensitivity as compared with the slab gel. Namely, such problems result from influence by Joulean heat that a band spreads and a temperature gradient takes place applying a high voltage in the slab gel, while such problems are less in the capillary column and spreading of a band is small to allow high sensitivity detection even if making high-speed migration while applying a high voltage.
When making electrophoresis employing a plurality of capillary columns, it is desirable that attachment/detachment of the capillary columns to/from an electrophoretic apparatus body is easy, and it is desirable to cassette the same with a holder for that In consideration of the recent environmental problem, it is also preferable that the capillary columns can be readily separated from the holder when discarded after use.
Furthermore, since a step by pressurization or decompression is requisite for charging gels into the capillary columns, airtightness is necessary for fixation of the capillary columns to the holder at least on sample injection sides in order to simultaneously charge the gels into all capillary columns contained in the cassette.
Accordingly, a first objective of the present invention is to provide a capillary cassette which can readily separate capillary columns from a holder and has sufficient airtightness between the capillary columns and the holder on sample injection sides.
The capillary columns are so thin that the same are difficult to manually work and are easily breakable. When fixing the capillary columns to the holder, it is a difficult operation requiring skill to bundle the same in plural, and in addition, it requires a long time.
Accordingly, a second objective of the present invention is to make it possible to simply and quickly perform preparation of a capillary cassette.
The capillary columns are protected with various coats, in order to increase mechanical strength. For coats, polyimide, silicone resin, polytetrafluoroethylene, acrylic resin and the like are used. When using the same as coats for capillary columns of electrophoresis however, optical characteristics of the coats such as light absorption and fluorescence hinder detection since optical means for absorbance measurement and fluorescence measurement are employed for detection of samples migrating in the capillary columns. Therefore, when the coats are made of materials hindering optical detection, the coats of detection parts are burned by flames and removed with burning means such as a lighter or a gas burner.
A multi-capillary electrophoretic apparatus using a plurality of capillary columns and simultaneously detecting a plurality of samples aligns, arranges and fixes such capillary columns that coats of detection parts are removed, thereby forming detection windows when the coats are made of materials hindering optical detection.
While a number of capillary columns are arranged in the multi-capillary electrophoretic apparatus, it follows that when performing coat removal of the detection parts on every one of the capillary columns, a long time is required for the operation.
Furthermore, the coating materials include such a one that a part located in a flame is completely burned and removed in the process of removing the coating material by burning by the flame while the same is melted but does not come to be removed in a portion separate from the flame and convex parts resulting from solidification of the melted coat are formed on both sides of the part from which the coat is removed. If such convex portions are formed, adjacent capillary columns do not adhere to each other or come to different levels due to the convex portions and detection windows are not planarly aligned when arranging the capillary columns, aligning positions of the parts from which the coats are removed and forming the detection windows. Consequently, the focus of an optical system may so deviate in detection that it cannot perform proper detection.
Accordingly, a third objective of the present invention is, when a coat for a capillary column is made of a material hindering optical detection, to provide such a capillary cassette that a detection window of a capillary column employed for a multi-capillary electrophoretic apparatus is planarly formed to be suitable for optical measurement and a method of manufacturing a capillary cassette having a feature in a detection window forming step thereof.
An initial capillary electrophoretic apparatus is that employing a single capillary column. In this case, it dips an end of the capillary column into a gel solution in a vessel, closes the vessel and pressurizes the vessel thereby pushing the gel into the capillary column and charging the same as a method of charging the gel into the capillary column. As another method, an operation of dipping an end of the capillary column into the gel solution, decompressing another end of the capillary column and sucking the gel into the capillary column, thereby charging the same, is also performed.
In multi-capillary electrophoresis, it is necessary to align capillary column end surfaces on sample injection sides in sample injection while it is necessary to align and handle capillary coat removing parts on detection sides when performing detection for handling a plurality of capillary columns, and hence it is desirable to provide such a capillary cassette that arrangement is fixed by a holder so that both the sample injection sides and the detection sides are in prescribed arrangement relation.
Furthermore, it is extremely troublesome to charge a gel into every one of the capillary columns in the state of the capillary cassette, and, in actuality, impossible in practice. Therefore, awaited is means for making it possible to simply perform gel charging into all capillary columns included therein in units of the capillary cassette.
Accordingly, a fourth objective of the present invention is to provide an apparatus to make it possible to simply charge gels into all capillary columns included in such a capillary cassette.
In order to attain the first objective, the present invention is such that, in a capillary cassette two-dimensionally arranging and fixing a plurality of capillary columns used in a multi-capillary electrophoretic apparatus on sample injection sides by a holder while arranging the same in a line on a plane on detection sides (sides opposite to the sample injection sides), the holder comprises a rubber plate so that end portions of the plurality of capillary columns pass through the rubber plate one by one to be fixed by elastic force of the rubber plate.
By inserting the capillary columns into the rubber plate and fixing the same with the holder on the sample injection sides, airtightness between the rubber plate and the capillary columns can be maintained and it is possible to serve a sealing function with respect to pressurization or suction in gel charging into capillaries. The capillary columns are not fixed by an adhesive or the like, whereby the capillary columns can be readily extracted from the holder in disposal of the capillary columns or the like.
In the present invention, it is preferable to comprise another holder pressing and holding the capillary columns from at least single surface sides of the plurality of capillary columns arranged in a line on the plane through the rubber plate on the detection sides of the capillary columns. By thus making fixation on the detection sides by pushing the capillary columns through the rubber plate, it is easy to detach the capillary columns from the holder on the detection sides, and an operation of separating the capillary columns and the holder in disposal after use or exchanging defective capillary columns becomes easy.
Furthermore, the holder on the detection sides preferably comprises clamping means fixing the capillary columns on both side portions while pressing and fixing the same on at least two portions of an intermediate part as means fixing the plurality of capillary columns arranged in a line on the plane through the rubber plate. By making it possible to press the capillary columns also in at least two portions of the intermediate part, it becomes easy to maintain arrangement on the plane.
A capillary cassette preparation method according to the present invention to attain the second objective is a method of passing through a supporter of a prescribed fixing position of capillary column fixing means having the supporter consisting of a rubber plate with a needle having an inner diameter larger than a capillary column from its vertical direction, guiding the capillary column into the needle and making the same pass through the supporter, thereafter extracting only the needle thereby holding and fixing the capillary column by the supporter and repeating this operation while changing the fixing position on the supporter thereby successively two-dimensionally arranging and fixing a plurality of capillary columns.
The capillary column guided in the needle to pass through the supporter of the rubber plate on a sample injection side holder (capillary column fixing means) is held and fixed by elastic force of rubber after only the needle is extracted from the supporter.
A capillary cassette preparation apparatus according to the present invention for executing this capillary cassette preparation method comprises arrangement position decision means holding capillary column fixing means having a supporter consisting of a rubber plate for moving and fixing the same in an in-plane direction of the supporter, a needle passing through the supporter comprised in the capillary column fixing means from a vertical direction of its plane and guiding a capillary column to the fixing position, a first capillary column holder holding the capillary column and inserting the same into the needle, a guide guiding the forward end of the capillary column held by the first capillary column into the needle, slide guide means moving the needle, the guide and the first capillary column holder in a rectilinear direction perpendicular to the plane of the supporter, a second capillary column holder holding the forward end portion of the capillary column made to project from the forward end of the needle in such a state that the needle passes through the supporter of the capillary column fixing means, cut means cutting the capillary column in a prescribed length, and a roller unit successively feeding the capillary column in an insert direction.
The fixing position of the capillary column is decided by mounting the capillary column fixing means on the arrangement position decision means and moving the arrangement position decision means upward, downward, leftward and rightward. It linearly moves the needle in the capillary column fixing means direction along the slide guide means and makes the same pass through the supporter on the fixing position. The forward end of the capillary column wound on a capillary drum is guided into the needle by the guide following the needle along the slide guide means and the first capillary column holder, and projects from the forward end of the needle. It holds the forward end portion of the capillary column by the second capillary column holder comprised on a needle projection side of the arrangement position decision means, and extracts only the needle from the supporter of the capillary column fixing means. The capillary column forward end portion remains in the supporter, elastic force of rubber which is the supporter acts in a direction closing a hole opened by the needle, and the capillary column is fixed to the capillary column fixing means. It cuts the fixed capillary column in the prescribed length by the cut means. The arrangement position decision means moves and brings a next fixing position onto a moving straight line of the needle, the guide and the capillary column holder. The capillary column wound on the drum is timely fed by the roller unit having a motor for motive power.
According to this capillary cassette preparation apparatus, capillary columns of a predetermined length are aligned in determined order by operating a lever in accordance with a prescribed procedure so that a capillary cassette can be prepared, whereby the efficiency of a preparation operation for the capillary cassette remarkably rises. Furthermore, it can simply make transition to automatization by mounting a driving source, whereby simple preparation of a capillary cassette with less failure is enabled.
In order to also form detection windows simultaneously with capillary cassette preparation, it is preferable to further comprise detection window preparation means removing capillary column coats of prescribed positions from the forward ends of the capillary columns and preparing the detection windows and arrangement means arranging terminating end sides of the cut capillary columns in a line in determined order in the capillary cassette preparation apparatus.
After the capillary columns are guided in the needle and pass through the capillary column fixing means, a voltage is applied to the detection window preparation means to prepare the detection windows on prescribed positions from the forward ends of the capillary columns. Thereafter the terminating ends of the capillary columns cut in the prescribed length are successively dropped between planes and arranged in a line by the arrangement means formed by two planes having a parallel clearance slightly wider than the capillary column outer diameter.
A capillary cassette according to the present invention for attaining the third object is such a one that a plurality of capillary columns are fixed by a holder on sample injection sides and planarly arranged in a line on detection sides, coats of the capillary columns are removed so that strip detection windows extending in the capillary column arrangement direction are formed on the capillary column arrangement on the detection sides, the capillary columns adhere to each other and the coats fuse to each other so that the capillary column arrangement is integrated around the detection windows.
In this capillary cassette, the capillary columns adhere to each other, the coats fuse to each other and the capillary column arrangement is integrated around the detection windows, whereby the detection windows planarly align, the focus of an optical system in detection does not deviate, and it is possible to perform correct detection.
When manufacturing the capillary cassette, a manufacturing method according to the present invention forms the detection windows including the following steps (A) and (B):
(A) a step of fixing the sample injection sides of the capillary columns using the holder and thereafter arranging and holding the capillary columns in a line planarly in close contact with each other, and
(B) a step of bringing heating means having a length for a plurality of capillary column outer diameters into contact with or approximating the same to a detection window formation planned region of the held capillary column arrangement for removing coats of the plurality of capillary columns while melting the coats around regions from which the coats are removed and thereafter solidifying the same for making the coats of the adjacent capillary columns fuse to each other.
It planarly arranges and fixes the capillary columns not subjected to coat removal of detection parts in a line and simultaneously performs coat removal of detection window formation planned positions of the plurality of capillary columns with the heating means. By simultaneously heating and removing the coats of the plurality of capillary columns, the adjacent capillary columns are fused and integrated with each other on positions separate from the regions from which the coats are removed when the melted coats are cooled and solidified. Thus, the plurality of capillary columns arranged in a line form a flat cable.
For a means of heating, a nichrome wire heater or a ceramic heater can be employed.
When using as the heating means employed for removing the coats that whose length is shorter than the width of the capillary column arrangement on the detection sides, it repeats the step (B) a number of times in the width direction of the capillary column arrangement for forming the detection windows.
This manufacturing method planarly arranges the plurality of columns and thereafter simultaneously removes the coats of the detection parts of the plurality of capillary columns, whereby remarkable reduction of the detection window preparation time can be made as compared with the case of removing the coat as to every single capillary collar.
In the peripheral parts of the detection windows formed by this manufacturing method, the coats melted but not come to be removed fuse the adjacent capillary columns when cooled and solidified. The plurality of fused capillary columns are previously planarly arranged and hence come to planarly maintain the detection windows after fusion, and it is possible to readily manufacture a capillary cassette capable of performing correct optical detection.
When fixing the detection sides of the plurality of capillary columns, it saves trouble of fixing the same one by one while it does not fix the same with an adhesive, whereby a time for drying the adhesive can also be saved.
A gel charging apparatus according to the present invention for attaining the fourth object is an apparatus for charging gels into capillary columns of a capillary cassette. In the capillary cassette, end portions of sample injection sides of a plurality of capillary columns mounted on a multi-capillary electrophoretic apparatus are two-dimensionally arranged while passing through a holding member of a holder and fixed while keeping airtightness between the same and the holding member.
Gel charging into the capillary columns can be performed either by suction or pressurization. In gel charging, it does not seal the respective capillary columns one by one to perform suction or pressurization but seals all capillary columns of the capillary cassette by the holder fixing the capillary columns while keeping airtightness to simultaneously perform gel charging.
In the system performing gel charging by suction, the present invention comprises a chamber provided with an opening in its upper surface so that closure means introducing end portions on sample injection sides of the capillary columns inside and closing the opening with the holder is provided in the opening and further provided with an exhaust port and a release port to the atmosphere, a gel vessel storing a gel solution so that end portions of the capillary columns opposite to the sample injection sides are dipped therein, exhaust means provided on the exhaust port of the chamber, and a switching valve provided on the release port.
When performing gel charging by suction, it dips the forward ends of the detection sides of all capillary columns of the capillary cassette in gels contained in the gel vessel, closes the opening of the chamber with the holder for the capillary cassette, decompresses the chamber with the exhaust means and inhales the gel solution into the capillary columns.
In the system performing gel charging by pressurization, the present invention comprises a chamber provided with an opening on its upper surface so that closure means introducing end portions of the sample injection sides of the capillary columns inside and closing the opening with the holder is provided in the opening while storing a gel solution on such a position that the end portions on the sample injection sides of the capillary columns are dipped therein in such a state that the opening is closed with the holder and further provided with a pressurization port and a release port to the atmosphere, pressurization means provided on the pressurization port of the chamber and a switching valve provided on the release port.
When performing gel charging by pressurization, it introduces the gel solution into the chamber, dips the forward ends of the capillary columns on the sample injection sides in the gel solution, closes the opening of the chamber with the holder, pressurizes the chamber with the pressurization means and pressure-fits gels into the capillary columns.
When charging the gel solution into the plurality of capillary columns forming a capillary array mounted on a multi-capillary electrophoretic apparatus, it does not directly close/fix the respective capillary columns but seals the plurality of capillary columns to the closure means with the sample injection side holder fixing the same with excellent airtightness to perform charging of the gels, whereby gel charging for the plurality of capillary columns can be simultaneously performed. Furthermore, simple mounting and airtightness in mounting can be compatibly attained.
Charging of the gel solution into the plurality of capillary columns can be simultaneously performed, whereby it does not damage the maximum merit of improvement of throughput by simultaneous performance of migration of a plurality of samples in multi-capillary electrophoresis in the process of gel preparation which is the pretreatment thereof.
It is preferable that a flow control valve controlling the flow rate is provided between the release port of the chamber and the switching valve. While the pressure in the chamber is released to the atmosphere after gel charging into the capillary columns, the speed at which the internal pressure of the chamber returns can be controlled with the flow control valve. While air may be mixed into the gels charged into the capillary columns when the degree of decompression or pressurization in the chamber is large, the degree of decompression or pressurization can be controlled with the flow control valve in this case.
In the system performing gel charging by pressurization, the gel solution may be directly introduced into the chamber, while it is preferable in the aspect of maintenance of the chamber when providing an attachable/detachable vessel in the chamber for storing the gel solution in the vessel.